Hepatitis delta virus (HDV) is a significant human pathogen that causes serious acute and chronic liver disease. There is no effective licensed therapy for this virus, which has a unique replication cycle that is not fully understood. HDV is a RNA virus that encodes a single protein, HDAg, that functions as a nucleoprotein; it forms ribonucleoprotein complexes with the viral (-) and (+) RNAs. These complexes play essential roles in replication of the viral RNA by host RNA polymerase but they are not well characterized because it has been difficult to assemble them in the laboratory. Recent developments have shown that it is now possible to create discrete RNP complexes in vitro by using HDAg from a genotype 3 strain of HDV. This proposal aims to take advantage of this recent development and to more fully characterize the HDV RNPs. There are two aims, each with sub-aims. Using microinjection techniques, the first sub-aims of Aim 1 will determine how many HDAg multimers must assemble on the RNA in order to initiate synthesis - first of the entire RNA replication cycle, then of the individual RNAs independently. The third part of Aim 1 will use the information gleaned from the first two sub-aims to rationally design an attempt to observe de novo HDV RNA transcription by Pol II in vitro. Aim 2 will determine aspects of the structure of the RNPs, with a particular focus on those RNPs shown in Aim 1 to be functional for RNA replication. One sub-aim will employ atomic force microscopy to visualize individually RNP complexes to see how the protein is distributed along the RNA and whether separate protein multimers interact with each other. The other two sub-aims will determine what specific sequences of the RNA are closely associated with HDAg in RNPs from cells, virions and from those assembled in vitro. Altogether, the proposal will substantially expand our understanding of the structure-function relationships in the HDV RNPs and how the virus coopts the host RNA polymerase for its replication.